Antigen screening system

ABSTRACT

Methods and compositions for identifying antigens of human lymphocytes are provided herein.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is copending with, shares at least one commoninventor with and claims priority to U.S. provisional patent applicationSer. No. 61/077,323, filed Jul. 1, 2008, the entire contents of whichare incorporated herein by reference.

BACKGROUND OF THE INVENTION

Despite advances in vaccine development and antimicrobial therapies,infectious diseases remain widespread. Nationwide, at least 45 millionpeople ages 12 and older, or one out of five adolescents and adults,have had genital Herpes infection. The prevalence of herpes simplexvirus-2 (HSV-2) is increasing worldwide and there is now evidencelinking it to HIV transmission. HIV has infected 40 million people todate and causes 2-3 million deaths each year (Joint United NationsProgramme on HIV/AIDS, 2006 Report on the Global AIDS Epidemic, Ch. 2,2006). Nine million new cases of tuberculosis were reported in 2005,with 1.6 million deaths in that year alone (Young et al., Clin Invest.118(4):1255-1265, 2008). Malaria causes disease in hundreds of millionsand kills 1-3 million each year (Snow et al., Nature 434 (7030):214-7,2005). Efforts to limit the spread of these and other pathogens andcontrol their devastating effects on human populations require effectivevaccines. There remains a need for improved vaccines, and for systems todevelop them.

The efficacy of many current vaccines lies in their ability to elicitrobust antibody responses. However, T cell mediated immunity isimportant for protection from many types of infections, including thosefor which no effective vaccine is available. CD4⁺ T cells orchestratehumoral and cell mediated immune responses in vivo. CD8⁺ T cells arecritical for the control of intracellular pathogens. Identifyingantigens that elicit T cell mediated responses has been more technicallylaborious than characterizing humoral antigens.

SUMMARY OF THE INVENTION

The present invention features, inter alia, methods of identifyingantigens of human lymphocytes as well as compositions including theantigens and methods of using the antigens. The invention also featuresmethods of evaluating an immune response in a human subject, e.g., fordiagnostic applications.

Accordingly, in certain embodiments, the present invention providesmethods of identifying antigens. In certain embodiments, methods include(a) providing a library of cells (e.g., bacterial cells), wherein eachcell of the library includes a heterologous polypeptide; (b) contactingthe cells with a first plurality of human cells which comprises humanantigen presenting cells that internalize library cells; (c) contactingthe first plurality of human cells with a second plurality of humancells, which second plurality includes human lymphocytes (e.g., T cells,such as CD4⁺ T cells, CD8⁺ T cells), under conditions in which thelymphocytes are stimulated by polypeptides presented by the firstplurality of human cells; and (d) determining whether a lymphocyte ofthe second plurality of human cells is stimulated by a heterologouspolypeptide presented by a cell of the first plurality of human cells.Stimulation of a lymphocyte by a heterologous polypeptide indicates thatthe heterologous polypeptide is an antigen.

In some embodiments, library cells include a cytolysin polypeptide, suchas listeriolysin O (LLO). In some embodiments, cells of the first andsecond plurality include primary cells (e.g., primary peripheral bloodmononuclear cells (PBMC)). In some embodiments, cells of the first andsecond plurality include cells from the same individual. In someembodiments, the individual is an individual that has been exposed to aninfectious agent, and the heterologous polypeptides are polypeptidesencoded by the infectious agent or a related species thereof. In someembodiments, the individual is an individual that has or had a cancer,or an autoimmune disease.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawing, and from the claims. All cited patents, andpatent applications and references (including references to publicsequence database entries) are incorporated by reference in theirentireties for all purposes.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph showing IFNγ secreted in supernatants by human CD8⁺ Tcells cocultured with autologous antigen presenting cells pulsed with E.coli expressing cytoplasmic listeriolysin O and polypeptides encoded byherpes simplex virus-2 (HSV-2) (single viral clones). Each barrepresents IFNγ levels secreted by T cells exposed to antigen presentingcells that had internalized library cells expressing a single viralpolypeptide. Black bars represent clones that had also been previouslyidentified by other methods.

DEFINITIONS

Antigen: The term “antigen”, as used herein, refers to a molecule (e.g.,a polypeptide) that elicits a specific immune response. Antigen specificimmunological responses, also known as adaptive immune responses, aremediated by lymphocytes (e.g., T cells, B cells) that express antigenreceptors (e.g., T cell receptors, B cell receptors). In certainembodiments, an antigen is a T cell antigen, and elicits a cellularimmune response. In certain embodiments, an antigen is a B cell antigen,and elicits a humoral (i.e., antibody) response. In certain embodiments,an antigen is both a T cell antigen and a B cell antigen. As usedherein, the term “antigen” encompasses both a full length polypeptide aswell as a portion of the polypeptide, and a peptide epitope within thepolypeptides (e.g., a peptide epitope bound by a MajorHistocompatibility Complex (MHC) molecule (e.g., MHC class I, or MHCclass II).

Antigen presenting cell: An “antigen presenting cell” or “APC” refers toa cell that presents peptides on MHC class I and/or MHC class IImolecules. APC include both professional APC (e.g., dendritic cells,macrophages, B cells), which have the ability to stimulate naïvelymphocytes, and non-professional APC (e.g., fibroblasts, epithelialcells, endothelial cells, glial cells). In certain embodiments, APC areable to internalize (e.g., endocytose) members of a library (e.g., cellsof a library of bacterial cells) that express heterologous polypeptidesas candidate antigens.

Autolysin polypeptide: An “autolysin polypeptide” is a polypeptide thatfacilitates or mediates autolysis of a cell (e.g., a bacterial cell)that has been internalized by a eukaryotic cell. In some embodiments, anautolysin polypeptide is a bacterial autolysin polypeptide. Autolysinpolypeptides include, and are not limited to, polypeptides whosesequences are disclosed in GenBank® under Acc. Nos. NP_388823.1,NP_266427.1, and P0AGC3.1.

Cytolysin polypeptide: A “cytolysin polypeptide” is a polypeptide thathas the ability to form pores in a membrane of a eukaryotic cell. Acytolysin polypeptide, when expressed in host cell (e.g., a bacterialcell) that has been internalized by a eukaryotic cell, facilitatesrelease of host cell components (e.g., host cell macromolecules, such ashost cell polypeptides) into the cytosol of the internalizing cell. Insome embodiments, a cytolysin polypeptide is bacterial cytolysinpolypeptide. In some embodiments, a cytolysin polypeptide is acytoplasmic cytolysin polypeptide. Cytolysin polypeptides include, andare not limited to, polypeptides whose sequences are disclosed in U.S.Pat. No. 6,004,815, and in GenBank® under Acc. Nos. NP_463733.1,NP_979614, NP_834769, YP_084586, YP_895748, YP_694620, YP_012823,NP_346351, YP_597752, BAB41212.2, NP_561079.1, and YP_001198769.

Cytoplasmic cytolysin polypeptide: A “cytoplasmic cytolysin polypeptide”is a cytolysin polypeptide that has the ability to form pores in amembrane of a cukaryotic cell, and that is expressed as a cytoplasmicpolypeptide in a bacterial cell. A cytoplasmic cytolysin polypeptide isnot significantly secreted by a bacterial cell. Cytoplasmic cytolysinpolypeptides can be provided by a variety of means. In some embodiments,a cytoplasmic cytolysin polypeptide has a sequence that is alteredrelative to the sequence of a secreted cytolysin polypeptide (e.g.,altered by deletion or alteration of a signal sequence to render itnonfunctional). In some embodiments, a cytoplasmic cytolysin polypeptideis cytoplasmic because it is expressed in a secretion-incompetent cell.In some embodiments, a cytoplasmic cytolysin polypeptide is cytoplasmicbecause it is expressed in a cell that does not recognize and mediatesecretion of a signal sequence linked to the cytolysin polypeptide. Insome embodiments, a cytoplasmic cytolysin polypeptide is a bacterialcytolysin polypeptide.

Heterologous: The term “heterologous”, as used herein to refer to genesor polypeptides, refers to a gene or polypeptide that does not naturallyoccur in the organism in which it is present and/or being expressed,and/or that has been introduced into the organism by the hand of man.Heterologous polypeptides used in accordance with the present inventionare typically polypeptides from infectious agents. In some embodiments,a heterologous polypeptide expressed in a bacterial host cell is apolypeptide encoded by a bacterial species that is different from thebacterial host cell species. In some embodiments, a heterologouspolypeptide expressed in a bacterial host cell is a polypeptide encodedby a virus. Where a plurality of different heterologous polypeptides areto be introduced into and/or expressed by cells (e.g., a library ofcells), the different polypeptides may all be from the same infectiousagent (e.g., from a single bacterial or viral species). In someembodiments, the different polypeptides are from more than oneinfectious agent (e.g., from two bacterial species, two viral species,or a bacterial species and a viral species). In some embodiments, theplurality of different polypeptides represents polypeptides from acomplete set of open reading frames (ORFs) expressed by an infectiousagent.

Invasin polypeptide: An “invasin polypeptide” is a polypeptide thatfacilitates or mediates uptake of a cell (e.g., a bacterial cell) by aeukaryotic cell. Expression of an invasin polypeptide in a noninvasivebacterial cell confers on the cell the ability to enter a eukaryoticcell. In some embodiments, an invasin polypeptide is a bacterial invasinpolypeptide. In some embodiments, an invasin polypeptide is a Yersiniainvasin polypeptide (e.g., a Yersinia invasin polypeptide comprising asequence disclosed in GenBank® under Acc. No. YP_070195.1).

Listeriolysin O (LLO): The terms “listeriolysin O” or “LLO” refer to alisteriolysin O polypeptide of Listeria monocytogenes and truncatedforms thereof that retain pore-forming ability (e.g., cytoplasmic formsof LLO, including truncated forms lacking a signal sequence). In someembodiments, an LLO is a cytoplasmic LLO. Exemplary LLO sequences areshown in Table 1, below.

Polypeptide: The term “polypeptide”, as used herein, generally has itsart-recognized meaning of a polymer of at least three amino acids. Thoseof ordinary skill in the art will appreciate, however, that the term“polypeptide” is intended to be sufficiently general as to encompass notonly polypeptides having the complete sequence recited herein (or in areference or database specifically mentioned herein), but also toencompass polypeptides that represent functional fragments (i.e.,fragments retaining at least one activity) of such completepolypeptides. Moreover, those of ordinary skill in the art understandthat protein sequences generally tolerate some substitution withoutdestroying activity. Thus, any polypeptide that retains activity andshares at least about 30-40% overall sequence identity, often greaterthan about 50%, 60%, 70%, or 80%, and further usually including at leastone region of much higher identity, often greater than 90% or even 95%,96%, 97%, 98%, or 99% in one or more highly conserved regions, usuallyencompassing at least 3-4 and often up to 20 or more amino acids, withanother polypeptide of the same class, is encompassed within therelevant term “polypeptide” as used herein. Other regions of similarityand/or identity can be determined by those of ordinary skill in the artby analysis of the sequences of various polypeptides.

Primary cells: As used herein, “primary cells” refers to cells from anorganism that have not been immortalized in vitro. In some embodiments,primary cells are cells taken directly from a subject (e.g., a human).In some embodiments, primary cells are progeny of cells taken from asubject (e.g., cells that have been passaged in vitro). Primary cellsinclude cells that have been stimulated to proliferate in culture.

Related species: As used herein, a species is “related” to anotherspecies if it is in the same family or genus and/or if antigens from thespecies elicit a cross-reactive immune response in an individual. Insome embodiments, cells from an individual that has been exposed to aninfectious agent are used to identify an antigen from the infectiousagent or a related species thereof. For example, cells from anindividual that has been exposed to a first herpesvirus species can bescreened against cells expressing candidate antigens from the firstherpesvirus species, or a second, related herpesvirus species.

Stimulate: As used herein, a peptide presented by an antigen presentingcell “stimulates” a lymphocyte if the lymphocyte is detectably activatedafter exposure to the peptide/APC under conditions that permit antigenspecific recognition to occur. Any indicator of lymphocyte activationcan be evaluated to determine whether a lymphocyte is stimulated (e.g.,proliferation, cytokine secretion, change in expression of one or moreactivation markers).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

The present invention provides systems for the rapid identification ofantigens that elicit T cell immunity, and particularly that elicit humanT cell immunity. The present invention allows analysis of completeproteomes of infectious agents, and is particularly powerful now thatcomplete genomes are available for hundreds of such infectious agents.The present invention therefore solves the ultimate challenge in vaccinedevelopment: identification of antigens that elicit T cell immunity inhumans.

Library Generation

A library is a collection of members (e.g., cells or non-cellularparticles, such as virus particles, liposomes, or beads (e.g., beadscoated with polypeptides, such as in vitro translated polypeptides,e.g., affinity beads, e.g., antibody coated beads, or NTA-Ni beads boundto polypeptides of interest). According to the present invention,members of a library include (e.g., internally express) heterologouspolypeptides. In some embodiments, members of a library which are cellsinternally express heterologous polypeptides. In some embodiments,members of a library which are particles include internally, and/or arebound to, heterologous polypeptides. Use of a library in an assay systemallows simultaneous evaluation in vitro of cellular responses tomultiple candidate antigens. Members of the library are constructed toinclude (e.g., internally express) heterologous polypeptides from aninfectious agent or target cell of interest (e.g., a tumor cell, or acell which is a target of an autoimmune response). According to thepresent invention, a library is designed to be internalized by humanantigen presenting cells so that peptides from library members,including peptides from internally expressed heterologous polypeptides,are presented on MHC molecules of the antigen presenting cells forrecognition by T cells.

Libraries can be used in assays that detect peptides presented by humanMHC class I and MHC class II molecules. Polypeptides expressed by theinternalized library members are digested in intracellular endocyticcompartments (e.g., phagosomes, endosomes, lysosomes) of the human cellsand presented on MHC class II molecules, which are recognized by humanCD4⁺ T cells. In some embodiments, library members include a cytolysinpolypeptide, in addition to the heterologous polypeptide. In someembodiments, library members include an invasin polypeptide, in additionto the heterologous polypeptide. In some embodiments, library membersinclude an autolysin polypeptide, in addition to the heterologouspolypeptide. In some embodiments, library members are provided withcells that express a cytolysin polypeptide (i.e., the cytolysin andheterologous polypeptide are not expressed in the same cell, and anantigen presenting cell is exposed to members that include the cytolysinand members that include the heterologous polypeptide, such that theantigen presenting cell internalizes both, and such that the cytolysinfacilitates delivery of heterologous polypeptides to the MHC class Ipathway of the antigen presenting cell). A cytolysin polypeptide can beconstitutively expressed in a cell, or it can be under the control of aninducible expression system (e.g., an inducible promoter). In someembodiments, a cytolysin is expressed under the control of an induciblepromoter to minimize cytotoxicity to the cell that expresses thecytolysin.

Once internalized by a human cell, a cytolysin polypeptide perforatesintracellular compartments in the human cell, allowing polypeptidesexpressed by the library members to gain access to the cytosol of thehuman cell. Polypeptides released into the cytosol are presented on MHCclass I molecules, which are recognized by CD8⁺ T cells.

A library can be comprised of any type of cell or particle that can beinternalized by, and deliver a heterologous polypeptide (and a cytolysinpolypeptide, in applications where a cytolysin polypeptide is desirable)to, antigen presenting cells for use in methods described herein.Although the term “cell” is used throughout the present specification torefer to a library member, it is understood that, in some embodiments,the library member is a non-cellular particle, such as a virus particle,liposome, or bead. In some embodiments, members of the library includepolynucleotides that encode the heterologous polypeptide (and cytolysinpolypeptide), and can be induced to express the heterologous polypeptide(and cytolysin polypeptide) prior to, and/or during internalization byantigen presenting cells. In some embodiments, members of the libraryinclude bacterial cells. In certain embodiments, the library includesnonpathogenic, nonvirulent bacterial cells. Examples of bacteria for useas library members include E. coli, mycobactcria, Listeriamonocytogenes, Shigella flexneri, Bacillus subtilis, or Salmonella.

In some embodiments, members of the library include eukaryotic cells(e.g., yeast cells). In some embodiments, members of the library includeviruses (e.g., bacteriophages). In some embodiments, members of thelibrary include liposomes. Methods for preparing liposomes that includea cytolysin and other agents are described in Kyung-Dall et al., U.S.Pat. No. 5,643,599. In some embodiments, members of the library includebeads. Methods for preparing libraries comprised of beads are described,e.g., in Lam et al., Nature 354: 82-84, 1991, U.S. Pat. Nos. 5,510,240and 7,262,269, and references cited therein.

In certain embodiments, a library is constructed by cloningpolynucleotides encoding open reading frames of an infectious agent ortarget cell, or portions thereof, into vectors that express the ORFs incells of the library. The polynucleotides can be cloned by designingprimers that amplify the ORFs. Primers can be designed using availablesoftware, such as Primer3Plus (available the following URL:bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi; see Rozen andSkaletsky, In: Krawetz S, Misener S (eds) Bioinformatics Methods andProtocols: Methods in Molecular Biology. Humana Press, Totowa, N.J., pp.365-386, 2000). Other methods for designing primers are known to thoseof skill in the art. In some embodiments, primers are constructed so asto produce polypeptides that are truncated, and/or lack hydrophobicregions (e.g., signal sequences or transmembrane regions) to promoteefficient expression. The location of predicted signal sequences andpredicted signal sequence cleavage sites in a given ORF sequence can bedetermined using available software, see, e.g., Dyrløv et al., J. Mol.Biol., 340:783-795, 2004, and the following URL:cbs.dtu.dk/services/SignalP/). For example, if a signal sequence ispredicted to occur at the N-terminal 20 amino acids of a givenpolypeptide sequence, a primer is designed to anneal to a codingsequence downstream of the nucleotides encoding the N-terminal 20 aminoacids, such that the amplified sequence encodes a product lacking thissignal sequence.

Primers can also be designed to include sequences that facilitatesubsequent cloning steps. ORFs can be amplified directly from genomicDNA (e.g., genomic DNA of an infectious agent), or from polynucleotidesproduced by reverse transcription (RT-PCR) of mRNAs expressed by theinfectious agent. RT-PCR of mRNA is useful, e.g., when the genomicsequence of interest contains intronic regions. PCR-amplified ORFs arecloned into an appropriate vector, and size, sequence, and expression ofORFs can be verified prior to use in immunological assays.

In some embodiments, an ORF sequence is linked to a sequence encoding atag (e.g., an N-terminal or C-terminal epitope tag). Epitope tagsfacilitate purification of expressed ORFs, and can allow one to verifythat a given ORF is properly expressed in a library host cell, e.g.,prior to using the cell in a screen. Useful epitope tags include, forexample, a polyhistidine (His) tag, a V5 epitope tag from the P and Vprotein of paramyxovirus, a hemagglutinin (HA) tag, a myc tag, andothers. In some embodiments, an ORF sequence is fused to a sequenceencoding a tag which is a known antigenic epitope (e.g., an MHC class I-and/or MHC class II-restricted T cell epitope of a model antigen such asan ovalbumin), and which can be used to verify that an ORF sequence isexpressed and that the ORF-tag fusion polypeptide is processed andpresented in antigen presentation assays. In some embodiments a tagincludes a T cell epitope of a murine T cell (e.g., a murine T cellline). In some embodiments, an ORF sequence is linked to a tag thatfacilitates purification and a tag that is a known antigenic epitope.

Polynucleotides encoding the ORFs are cloned into an expression vectorfor introduction into library host cells. Various vector systems areavailable to facilitate cloning and manipulation of ORFs, such as theGateway® Cloning system (Invitrogen). As is known to those of skill inthe art, ORF expression vectors include elements that drive productionof polypeptides encoded by the ORF in library host cells (e.g., promoterand other regulatory elements). In some embodiments, ORF expression iscontrolled by an inducible element (e.g., an inducible promoter, e.g.,an IPTG- or arabinose-inducible promoter, or an IPTG-inducible phage T7RNA polymerase system, a lactose (lac) promoter, a tryptophan (trp)promoter, a tac promoter, a trc promoter, a phage lambda promoter, analkaline phosphatase (phoA) promoter, to give just a few examples; seeCantrell, Meth. in Mol. Biol., 235:257-276, Humana Press, Casali andPreston, Eds.). In some embodiments, ORFs are expressed as cytoplasmicpolypeptides. In some embodiments, the vector used for ORF expression isa vector that has a high copy number in a library host cell. In someembodiments, the vector used for expression has a copy number that ismore than 25, 50, 75, 100, 150, 200, or 250 copies per cell. In someembodiments, the vector used for expression has a ColE1 origin ofreplication. Useful vectors for polypeptide expression in bacteriainclude pET vectors (Novagen), Gateway® pDEST vectors (Invitrogen), pGEXvectors (Amersham Biosciences), pPRO vectors (BD Biosciences), pBADvectors (Invitrogen), pLEX vectors (Invitrogen), pMAL™ vectors (NewEngland BioLabs), pGEMEX vectors (Promega), and pQE vectors (Qiagen).Vector systems for producing phage libraries are known and includeNovagen T7Select® vectors, and New England Biolabs Ph.D.™ PeptideDisplay Cloning System.

In some embodiments, library host cells express (either constitutively,or when induced, depending on the selected expression system) an ORF toat least 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the total cellularprotein. In some embodiments, the level of expression of an ORF in alibrary member (e.g., cell, virus particle, liposome, bead) is such thatantigen presenting cells exposed to a sufficient quantity of the librarymembers present on MHC molecules ORF epitopes at a density that iscomparable to the density presented by antigen presenting cells pulsedwith purified peptides.

Methods for efficient, large-scale production of ORF libraries areavailable. For example, site-specific recombinases or rare-cuttingrestriction enzymes can be used to transfer ORFs between expressionvectors in the proper orientation and reading frame (Walhout et al.,Meth. Enzymol. 328:575-592, 2000; Marsischky et al., Genome Res.14:2020-202, 2004; Blommel et al., Protein Expr. Purif. 47:562-570,2006).

For production of liposome libraries, expressed polypeptides (e.g.,purified or partially purified polypeptides) can be entrapped inliposomal membranes, e.g., as described in Wassef et al., U.S. Pat. No.4,863,874; Wheatley et al., U.S. Pat. No. 4,921,757; Huang et al., U.S.Pat. No. 4,925,661; or Martin et al., U.S. Pat. No. 5,225,212.

A library can be designed to include full length polypeptides and/orportions of polypeptides encoded by an infectious agent or target cell.Expression of full length polypeptides maximizes epitopes available forpresentation by a human antigen presenting cell, thereby increasing thelikelihood of identifying an antigen. However, in some embodiments, itis useful to express portions of ORFs, or ORFs that are otherwisealtered, to achieve efficient expression. For example, in someembodiments, ORFs encoding polypeptides that are large (e.g., greaterthan 1,000 amino acids), that have extended hydrophobic regions, signalpeptides, transmembrane domains, or domains that cause cellulartoxicity, are modified (e.g., by C-terminal truncation, N-terminaltruncation, or internal deletion) to reduce cytotoxicity and permitefficient expression a library cell, which in turn facilitatespresentation of the encoded polypeptides on human cells. Other types ofmodifications, such as point mutations or codon optimization, may alsobe used to enhance expression.

The number of polypeptides included in a library can be varied. Alibrary can be designed to express polypeptides from at least 5%, 10%,15%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 98%, 99%, or more, of the ORFs in an infectious agent ortarget cell. In some embodiments, a library expresses at least 25, 50,75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, or 1000 different heterologous polypeptides,each of which may represent a polypeptide encoded by a single fulllength ORF or portion thereof.

In some embodiments, it is advantageous to include polypeptides from asmany ORFs as possible, to maximize the number of candidate antigens forscreening. In some embodiments, a subset of polypeptides having aparticular feature of interest is expressed. For example, for assaysfocused on identifying antigens associated with a particular stage ofinfection, one can construct a library that expresses a subset ofpolypeptides associated with that stage of infection (e.g., a librarythat expresses polypeptides associated with the hepatocyte phase ofinfection by Plasmodium falciparum, e.g., a library that expressespolypeptides associated with a yeast or mold stage of a dimorphic fungalpathogen). In some embodiments, assays may focus on identifying antigensthat are secreted polypeptides, cell surface-expressed polypeptides, orvirulence determinants, e.g., to identify antigens that are likely to betargets of both humoral and cell mediated immune responses.

In addition to heterologous polypeptides, libraries can include tagsthat allow one to easily purify, analyze, or evaluate MHC presentation,of the heterologous polypeptide. In some embodiments, ORFs expressed bya library include C-terminal tags that include both an MHC class I andan MHC class II-restricted T cell epitope from a model antigen, such aschicken ovalbumin (OVA). Library protein expression and MHC presentationis validated using these epitopes. In some embodiments, the epitopes areOVA₂₄₇₋₂₆₅ and OVA₂₅₈₋₂₆₅ respectfully, corresponding to positions inthe amino acid sequence found in GenBank® under Acc. No. NP_990483.Expression and presentation of linked ORFs can be verified with antigenpresentation assays using T cell hybridomas (e.g., B3Z T hybridomacells, which are H2-K^(b) restricted, and KZO T hybridoma cells, whichare H2-A^(k) restricted) that specifically recognize these epitopes (seeExample 3, below).

Methods and compositions described herein can be used to identify anddetect responses to antigens for any agent that infects humans. Forexample, libraries can be designed to express polypeptides encoded byviruses, bacteria, fungi, protozoa, or helminths that infect humans.

In some embodiments, members of a library include polynucleotides thatencode polypeptides from a virus. For example, a library can be designedto express polypeptides from one of the following viruses: animmunodeficiency virus (e.g., a human immunodeficiency virus (HIV),e.g., HIV-1, HIV-2), a hepatitis virus (e.g., hepatitis B virus (HBV),hepatitis C virus (HCV), hepatitis A virus, non-A and non-B hepatitisvirus), a herpes virus (e.g., herpes simplex virus type I (HSV-1),HSV-2, Varicella-zoster virus, Epstein Barr virus, humancytomegalovirus, human herpesvirus 6 (HHV-6), HHV-7, HHV-8), a poxvirus(e.g., variola, vaccinia, monkeypox, Molluscum contagiosum virus), aninfluenza virus, a human papilloma virus, adenovirus, rhinovirus,coronavirus, respiratory syncytial virus, rabies virus, coxsackie virus,human T-cell leukemia virus (types I, II and III), parainfluenza virus,paramyxovirus, poliovirus, rotavirus, rhinovirus, rubella virus, measlesvirus, mumps virus, adenovirus, yellow fever virus, Norwalk virus, WestNile virus, a Dengue virus, Severe Acute Respiratory SyndromeCoronavirus (SARS-CoV), bunyavirus, Ebola virus, Marburg virus, Easternequine encephalitis virus, Venezuelan equine encephalitis virus,Japanese encephalitis virus, St. Louis encephalitis virus, Junin virus,Lassa virus, and Lymphocytic choriomeningitis virus. Libraries for otherviruses can also be produced and used according to methods describedherein.

In some embodiments, members of a library include polynucleotides thatencode polypeptides from bacteria (e.g., from a bacterial pathogen). Insome embodiments, the bacterial pathogen is an intracellular pathogen.In some embodiments, the bacterial pathogen is an extracellularpathogen. Examples of bacterial pathogens include bacteria from thefollowing genera and species: Chlamydia (e.g., Chlamydia pneumoniae,Chlamydia psittaci, Chlamydia trachomatis), Legionella (e.g., Legionellapneumophila), Listeria (e.g., Listeria monocytogenes), Rickettsia (e.g.,R. australis, R. rickettsii, R. akari, R. conorii, R. sibirica, R.japonica, R. africae, R. typhi, R. prowazekii), Actinobacter (e.g.,Actinobacter baumannii), Bordetella (e.g., Bordetella pertussis),Bacillus (e.g., Bacillus anthracis, Bacillus cereus), Bacteroides (e.g.,Bacteroides fragilis), Bartonella (e.g., Bartonella henselae), Borrelia(e.g., Borrelia burgdorferi), Brucella (e.g., Brucella abortus, Brucellacanis, Brucella melitensis, Brucella suis), Campylobacter (e.g.,Campylobacter jejuni), Clostridium (e.g., Clostridium botulinum,Clostridium difficile, Clostridium perfringens, Clostridium tetani),Corynebacterium (e.g., Corynebacterium diphtheriae, Corynebacteriumamycolatum), Enterococcus (e.g., Enterococcus faecalis, Enterococcusfaecium), Escherichia (e.g., Escherichia coli), Francisella (e.g.,Francisella tularensis), Haemophilus (e.g., Haemophilus influenzae),Helicobacter (e.g., Helicobacter pylori), Klebsiella (e.g., Klebsiellapneumoniae), Leptospira (e.g., Leptospira interrogans), Mycobacteria(e.g., Mycobacterium leprae, Mycobacterium tuberculosis), Mycoplasma(e.g., Mycoplasma pneumoniae), Neisseria (e.g., Neisseria gonorrhoeae,Neisseria meningitidis), Pseudomonas (e.g., Pseudomonas aeruginosa),Salmonella (e.g., Salmonella typhi, Salmonella typhimurium, Salmonellaenterica), Shigella (e.g., Shigella dysenteriae, Shigella sonnei),Staphylococcus (e.g., Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus saprophyticus), Streptococcus (e.g., Streptococcusagalactiae, Streptococcus pneumoniae, Streptococcus pyogenes), Treponoma(e.g., Treponoma pallidum), Vibrio (e.g., Vibrio cholerae, Vibriovulnificus), and Yersinia (e.g., Yersinia pestis). Libraries for otherbacteria can also be produced and used according to methods describedherein.

In some embodiments, members of a library include polynucleotides thatencode polypeptides from protozoa. Examples of protozoal pathogensinclude the following organisms: Cryptosporidium parvum, Entamoeba(e.g., Entamoeba histolytica), Giardia (e.g., Giardia lambila),Leishmania (e.g., Leishmania donovani), Plasmodium spp. (e.g.,Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodiummalariae), Toxoplasma (e.g., Toxoplasma gondii), Trichomonas (e.g.,Trichomonas vaginalis), and Trypanosoma (e.g., Trypanosoma brucei,Trypanosoma cruzi). Libraries for other protozoa can also be producedand used according to methods described herein.

In some embodiments, members of a library include polynucleotides thatencode polypeptides from a fungus. Examples of fungal pathogens includethe following: Aspergillus, Candida (e.g., Candida albicans),Coccidiodes (e.g., Coccidiodes immitis), Cryptococcus (e.g.,Cryptococcus neoformans), Histoplasma (e.g., Histoplasma capsulatum),and Pneumocystis (e.g., Pneumocystis carinii). Libraries for other fungican also be produced and used according to methods described herein.

In some embodiments, members of a library include polynucleotides thatencode polypeptides from a helminth. Examples of helminthic pathogensinclude Ascaris lumbricoides, Ancylostomna, Clonorchis sinensis,Dracuncula mnedinensis, Enterobius vermicularis, Filaria, Onchocercavolvulus, Loa loa, Schistosoma, Strongyloides, Trichuris trichura, andTrichinella spiralis. Libraries for other helminths can also be producedand used according to methods described herein.

Sequence information for genomes and ORFs for infectious agents ispublicly available. See, e.g., the Entrez Genome Database (URL:ncbi.nlm.nih.gov/sites/entrez?db-Genome&itool=toolbar) and the ERGO™Database (URL:igwcb.integratcdgcnomics.com/ERGO_supplement/genomes.html), the GenomesOnline Database (GOLD)(URL: genomesonline.org)(Liolios et al., NucleicAcids Res. 1; 34(Database issue):D332-4, 2006).

In some embodiments, a library includes polynucleotides that expresshuman polypeptides. Such libraries are useful, e.g., for identifyingcandidate tumor antigens, or targets of autoreactive immune responses.An exemplary library for identifying tumor antigens includespolynucleotides encoding polypeptides that are differentially expressedor otherwise altered in tumor cells. An exemplary library for evaluatingautoreactive immune responses includes polynucleotides expressed in thetissue against which the autoreactive response is directed (e.g., alibrary containing pancreatic polynuclcotide sequences is used forevaluating an autoreactive immune response against the pancreas).

As noted above, library members can express a cytolysin polypeptide, inaddition to a heterologous polypeptide. In some embodiments, thecytolysin polypeptide is heterologous to the library cell in which it isexpressed, and facilitates delivery of polypeptides expressed by thelibrary cell into the cytosol of a human cell that has internalized thelibrary cell. Cytolysin polypeptides include bacterial cytolysinpolypeptides, such as listeriolysin O (LLO), streptolysin O (SLO), andperfringolysin O (PFO). Additional cytolysin polypeptides are describedin U.S. Pat. No. 6,004,815. In certain embodiments, library membersexpress LLO. In some embodiments, a cytolysin polypeptide is notsignificantly secreted by the library cell (e.g., less than 20%, 10%,5%, or 1% of the cytolysin polypeptide produced by the cell issecreted). For example, the cytolysin polypeptide is a cytoplasmiccytolysin polypeptide, such as a cytoplasmic LLO polypeptide (e.g., aform of LLO which lacks the N-terminal signal sequence, as described inHiggins et al., Mol. Microbiol. 31(6):1631-1641, 1999). Exemplarycytolysin polypeptide sequences are shown in Table 1. The listeriolysinO (Δ3-25) sequence shown in the second row of Table 1 has a deletion ofresidues 3-25, relative to the LLO sequence in shown in the first row ofTable 1, and is a cytoplasmic LLO polypeptide. In some embodiments, acytolysin is expressed constitutively in a library host cell. In otherembodiments, a cytolysin is expressed under the control of an induciblepromoter. Cytolysin polypeptides can be expressed from the same vector,or from a different vector, as the heterologous polypeptide in a librarycell.

TABLE 1 Exemplary Cytolysin Polypeptides Polypeptide Polypeptide NameAccession No. (species) GI No. Polypeptide Sequence listeriolysin ONP_463733.1 MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSMA (ListeriaGI:16802248 PPASPPASPKTPIEKKHADEIDKYIQGLDYNKNNVLVYHG monocytogenes)DAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYDPEGNEIVQHKNWSENNKSKLAHFTSSIYLPGNARNINVYAKECTGLAWEWWRTVIDDRNLPLVKNRNISIWGTTLYPKY SNKVDNPIE (SEQ ID NO:_)listeriolysin O MKDASAFNKENSISSMAPPASPPASPKTPIEKKHADEIDK (Δ3-25)YIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYDPEGNEIVQHKNWSENNKSKLAHFTSSIYLPGNARNINVYAKECTGLAWEWWRTVIDDRNLPLVKNRNISIWGTTLYPKYSNKVDNPIE (SEQ ID NO:_) streptolysin O BAB41212.2MSNKKTFKKYSRVAGLLTAALIIGNLVTANAESNKQNTAS (Streptococcus GI:71061060TETTTTSEQPKPESSELTIEKAGQKMDDMLNSNDMIKLAP pyogenes)KEMPLESAEKEEKKSEDKKKSEEDHTEEINDKIYSLNYNELEVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTPVDISIIDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGMGDKATVEVNDPTYANVSTAIDNLVNQWHDNYSGGNTLPARTQYTESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAYKQIFYTVSANLPNNPADVFDKSVTFKDLQRKGVSNEAPPLFVSNVAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDILENSSFTAVVLGGDAAEHNKVVTKDFDVIRNVIKDNATFSRKNPAYPISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQGAYVAQYEILWDEINYDDKGKEVITKRRWDNNWYSKTSPFSTVIPLGANSRNIRIMARECTGLAWEWWRKVIDERDVKLSKEINVNISGS TLSPYGSITYK (SEQ ID NO:_)perfringolysin O NP_561079.1 MIRFKKTKLIASIAMALCLFSQPVISFSKDITDKNQSIDS(Clostridium GI:18309145 GISSLSYNRNEVLASNGDKIESFVPKEGKKTGNKFIVVERperfringens) QKRSLTTSPVDISIIDSVNDRTYPGALQLADKAFVENRPTILMVKRKPININIDLPGLKGENSIKVDDPTYGKVSGAIDELVSKWNEKYSSTHTLPARTQYSESMVYSKSQISSALNVNAKVLENSLGVDFNAVANNEKKVMILAYKQIFYTVSADLPKNPSDLFDDSVTFNDLKQKGVSNEAPPLMVSNVAYGRTIYVKLETTSSSKDVQAAFKALIKNTDIKNSQQYKDIYENSSFTAVVLGGDAQEHNKVVTKDFDEIRKVIKDNATFSTKNPAYPISYTSVFLKDNSVAAVHNKTDYIETTSTEYSKGKINLDHSGAYVAQFEVAWDEVSYDKEGNEVLTHKTWDGNYQDKTAHYSTVIPLEANARNIRIKARECTGLAWEWWRDVISEYDVPLTNNINVSIWGTTLYPGSSITYN (SEQ ID NO:_)

In some embodiments, a library member (e.g., a library member which is abacterial cell) includes an invasin that facilitates uptake by theantigen presenting cell. In some embodiments, a library member includesan autolysin that facilitates autolysis of the library member within theantigen presenting cell. In some embodiments, a library member includesboth an invasin and an autolysin. In some embodiments, a library memberwhich is an E. coli cell includes an invasin and/or an autolysin. Invarious embodiments, library cells that express an invasin and/orautolysin are used in methods that also employ non-professional antigenpresenting cells or antigen presenting cells that are from cell lines.Isberg et al. (Cell, 1987, 50:769-778), Sizemore et al. (Science, 1995,270:299-302) and Courvalin et al. (C.R. Acad. Sci. Paris, 1995,318:1207-12) describe expression of an invasin to effect endocytosis ofbacteria by target cells. Autolysins are described by Cao et al.,Infect. Immun. 1998, 66(6): 2984-2986; Margot et al., J. Bacteriol.1998, 180(3):749-752; Buist et al., Appl. Environ. Microbiol., 1997,63(7):2722-2728; Yamanaka et al., FEMS Microbiol. Lett., 1997, 150(2):269-275; Romero et al., FEMS Microbiol. Lett., 1993, 108(1):87-92;Betzner and Keck, Mol. Gen. Genet., 1989, 219(3): 489-491; Lubitz etal., J. Bacteriol., 1984, 159(1):385-387; and Tomasz et al., J.Bacteriol., 1988, 170(12): 5931-5934. In some embodiments, an autolysinhas a feature that permits delayed lysis, e.g., the autolysin istemperature-sensitive or time-sensitive (see, e.g., Chang et al., 1995,J. Bact. 177, 3283-3294; Raab et al., 1985, J. Mol. Biol. 19, 95-105;Gerds et al., 1995, Mol. Microbiol. 17, 205-210). Useful cytolysins alsoinclude addiction (poison/antidote) autolysins, (see, e.g., Magnuson R,et al., 1996, J. Biol. Chem. 271(31), 18705-18710; Smith A S, et al.,1997, Mol. Microbiol. 26(5), 961-970).

In most embodiments, a member of a library expresses a singleheterologous polypeptide of an infectious agent or target cell. In someembodiments, a cell of a library expresses multiple heterologouspolypeptides. Sets of library members (e.g., bacterial cells) can beprovided on an array (e.g., on a solid support, such as a 96-well plate)and separated such that members in each location express a differentheterologous polypeptide, or a different set of heterologouspolypeptides.

Methods of using library members for identifying T cell antigens aredescribed in detail below. In addition to these methods, library membersalso have utility in assays to identify B cell antigens. For example,lysate prepared from library members that include heterologouspolypeptides can be used to screen a sample comprising antibodies (e.g.,a serum sample) from an individual (e.g., an individual that has beenexposed to an infectious agent of interest), to determine whetherantibodies present in the individual react with the heterologouspolypeptide. Suitable methods for evaluating antibody reactivity areknown and include ELISA assays.

Library Screens

Human Cells for Antigen Presentation

The present invention provides, inter alia, compositions and methods foridentifying antigens recognized by human immune cells. Human antigenpresenting cells express ligands for antigen receptors and other immuneactivation molecules on human lymphocytes. Given differences in MHCpeptide binding specificities and antigen processing enzymes betweenspecies, antigens processed and presented by human cells are more likelyto be physiologically relevant human antigens in vivo than antigensidentified in non-human systems. Accordingly, methods of identifyingthese antigens employ human cells to present candidate antigenpolypeptides.

Any human cell that internalizes library members and presentspolypeptides expressed by the library members on MHC molecules can beused as an antigen presenting cell according to the present invention.In some embodiments, human cells used for antigen presentation areprimary human cells. The cells can include peripheral blood mononuclearcells (PBMC) of a human. In some embodiments, peripheral blood cells areseparated into subsets (e.g., subsets comprising dendritic cells,macrophages, monocytes, B cells, or combinations thereof) prior to usein an antigen presentation assay. In some embodiments, a subset of cellsthat expresses MHC class II is selected from peripheral blood. In oneexample, a cell population including dendritic cells is isolated fromperipheral blood. In some embodiments, a subset of dendritic cells isisolated (e.g., plasmacytoid, myeloid, or a subset thereof). Humandendritic cell markers include CD1c, CD1a, CD303, CD304, CD141, andCD209. Cells can be selected based on expression of one or more of thesemarkers (e.g., cells that express CD303, CD1c, and CD141).

Dendritic cells can be isolated by positive selection from peripheralblood using commercially available kits (e.g., from Miltenyi BiotecInc.). Dendritic cells can also be produced by culturing peripheralblood cells under conditions that promote differentiation of monocyteprecursors into dendritic cells in vitro. These conditions typicallyinclude culturing the cells in the presence of cytokines such as GM-CSFand IL-4 (see, e.g., Inaba et al., Isolation of dendritic cells, Curr.Protoc. Immunol. May; Chapter 3: Unit 3.7, 2001). Procedures for invitro expansion of hematopoietic stem and progenitor cells (e.g., takenfrom bone marrow or peripheral blood), and differentiation of thesecells into dendritic cells in vitro, is described in U.S. Pat. No.5,199,942, and U.S. Pat. Pub. 20030077263. Briefly, CD34⁺ hematopoieticstem and progenitor cells are isolated from peripheral blood or bonemarrow and expanded in vitro in culture conditions that include one ormore of Flt3-L, IL-1, IL-3, and c-kit ligand.

In some embodiments, immortalized cells that express human MHC molecules(e.g., human cells, or non-human cells that are engineered to expresshuman MHC molecules) are used for antigen presentation. For example,assays can employ COS cells transfected with human MHC molecules or HeLacells.

In some embodiments, primary human cells are used in a method describedherein and both the antigen presenting cells and immune cells used inthe method are derived from the same subject (e.g., autologous T cellsand APC are used). In these embodiments, it can be advantageous tosequentially isolate subsets of cells from peripheral blood of thesubject, to maximize the yield of cells available for assays. Forexample, one can first isolate CD4⁺ and CD8⁺ T cell subsets from theperipheral blood. Next, dendritic cells (DC) are isolated from the Tcell-depleted cell population. The remaining T- and DC-depleted cellsare used to supplement the DC in assays, or are used alone as antigenpresenting cells. In some embodiments, DC are used with T- andDC-depleted cells in an assay, at a ratio of 1:2, 1:3, 1:4, or 1:5.

Antigen presenting cells can be isolated from sources other thanperipheral blood. For example, antigen presenting cells can be takenfrom a mucosal tissue (e.g., nose, mouth, bronchial tissue, trachealtissue, the gastrointestinal tract, the genital tract (e.g., vaginaltissue), or associated lymphoid tissue), peritoneal cavity, lymph nodes,spleen, bone marrow, thymus, lung, liver, kidney, neuronal tissue,endocrine tissue, or other tissue, for use in screening assays. In someembodiments, cells are taken from a tissue that is the site of an activeimmune response (e.g., an ulcer, sore, or abscess). Cells may beisolated from tissue removed surgically, via lavage, or other means.

Antigen presenting cells useful in methods described herein are notlimited to “professional” antigen presenting cells. Surprisingly, thepresent inventors have demonstrated that non-professional antigenpresenting cells can be utilized effectively in the practice of thepresent invention. Non-professional antigen presenting cells includefibroblasts, epithelial cells, endothelial cells, neuronal/glial cells,lymphoid or myeloid cells that are not professional antigen presentingcells (e.g., T cells, neutrophils), muscle cells, liver cells, and othertypes of cells.

Antigen presenting cells are cultured with library members that expressa heterologous polypeptide (and, if desired, a cytolysin polypeptide)under conditions in which the antigen presenting cells internalize,process and present polypeptides expressed by the library members on MHCmolecules. In some embodiments, library members are killed orinactivated prior to culture with the antigen presenting cells. Cells orviruses can be inactivated by any appropriate agent (e.g., fixation withorganic solvents, irradiation, freezing). In some embodiments, thelibrary members are cells that express ORFs linked to a tag (e.g., a tagwhich comprises one or more known T cell epitopes), expression of whichhas been verified prior to the culturing.

In some embodiments, antigen presenting cells are incubated with librarymembers at 37° C. for between 30 minutes and 5 hours (e.g., for 45 min.to 1.5 hours). After the incubation, the antigen presenting cells can bewashed to remove library members that have not been internalized. Incertain embodiments, the antigen presenting cells are non-adherent, andwashing requires centrifugation of the cells. The washed antigenpresenting cells can be incubated at 37° C. for an additional period oftime (e.g., 30 min. to 2 hours) prior to exposure to lymphocytes, toallow antigen processing. In some embodiments, it is desirable to fixand kill the antigen presenting cells prior to exposure to lymphocytes(e.g., by treating the cells with 1% paraformaldehyde).

The antigen presenting cell and library member numbers can be varied, solong as the library members provide quantities of heterologouspolypeptides sufficient for presentation on MHC molecules. In someembodiments, antigen presenting cells are provided in an array, and arecontacted with sets of library cells, each set expressing a differentheterologous polypeptide. In certain embodiments, each location in thearray includes 1×10³-1×10⁶ antigen presenting cells, and the cells arecontacted with 1×10³-1×10⁸ library cells which are bacterial cells.

In any of the embodiments described herein, antigen presenting cells canbe freshly isolated, maintained in culture, or thawed from frozenstorage prior to incubation with library cells, or after incubation withlibrary cells.

Human Lymphocytes

In methods of the present invention, human lymphocytes are tested forantigen specific reactivity to antigen presenting cells, e.g., antigenpresenting cells that have been incubated with libraries expressingcandidate antigen polypeptides as described above. The methods of thepresent invention permits rapid identification of human antigens usingpools of lymphocytes isolated from an individual, or progeny of thecells. The detection of antigen specific responses does not rely onlaborious procedures to isolate individual T cell clones. In someembodiments, the human lymphocytes are primary lymphocytes. Just asantigen presenting cells may be separated into subsets prior to use inantigen presentation assays, a population of lymphocytes having aspecific marker or other feature can be used. In some embodiments, apopulation of T lymphocytes is isolated. In some embodiments, apopulation of CD4⁺ T cells is isolated. In some embodiments, apopulation of CD8⁺ T cells is isolated. CD8⁺ T cells recognize peptideantigens presented in the context of MHC class I molecules. Thus, insome embodiments, the CD8⁺ T cells are used with antigen presentingcells that have been exposed to library host cells that co-express acytolysin polypeptide, in addition to a candidate antigen. T cellsubsets that express other cell surface markers may also be isolated,e.g., to provide cells having a particular phenotype. These include CLA(for skin-homing T cells), CD25, CD30, CD69, CD154 (for activated Tcells), CD45RO (for memory T cells), CD294 (for Th2 cells), γ/δTCR-expressing cells, CD3 and CD56 (for NK T cells). Other subsets canalso be selected.

Lymphocytes can be isolated, and separated, by any means known in theart (e.g., using antibody-based methods such as those that employmagnetic bead separation, panning, or flow cytometry). Reagents toidentify and isolate human lymphocytes and subsets thereof are wellknown and commercially available.

Lymphocytes for use in methods described herein can be isolated fromperipheral blood mononuclear cells, or from other tissues in a human. Insome embodiments, lymphocytes are taken from lymph nodes, a mucosaltissue (e.g., nose, mouth, bronchial tissue, tracheal tissue, thegastrointestinal tract, the genital tract (e.g., vaginal tissue), orassociated lymphoid tissue), peritoneal cavity, spleen, thymus, lung,liver, kidney, neuronal tissue, endocrine tissue, peritoneal cavity,bone marrow, or other tissues. In some embodiments, cells are taken froma tissue that is the site of an active immune response (e.g., an ulcer,sore, or abscess). Cells may be isolated from tissue removed surgically,via lavage, or other means.

Lymphocytes taken from an individual can be maintained in culture orfrozen until use in antigen presentation assays. Suprisingly, theinventors have found that freshly isolated lymphocytes can be stimulatedin vitro by antigen presenting cells exposed to library cells asdescribed above. These lymphocytes exhibit detectable stimulationwithout the need for prior non-antigen specific expansion. However,primary lymphocytes also elicit detectable antigen specific responseswhen first stimulated nonspecifically in vitro. Thus, in someembodiments, lymphocytes are stimulated to proliferate in vitro in a nonantigen-specific manner, prior to use in an antigen presentation assay.Lymphocytes can also be stimulated in an antigen-specific manner priorto use in an antigen presentation assay. For example, cells from anindividual thought to have been exposed to a virus can be stimulatedwith antigen presenting cells infected with the virus, or pulsed with acomposition comprising viral antigens (e.g., viral lysate, orrecombinant polypeptides). In some embodiments, cells are stimulated toproliferate by a library (e.g., prior to use in an antigen presentationassay that employs the library). Expanding cells in vitro providesgreater numbers of cells for use in assays. Primary T cells can bestimulated to expand, e.g., by exposure to a polyclonal T cell mitogen,such as phytohemagglutinin or concanavalin, by treatment with antibodiesthat stimulate proliferation, or by treatment with particles coated withthe antibodies. In some embodiments, T cells are expanded by treatmentwith anti-CD2, anti-CD3, and anti-CD28 antibodies.

Antigen Presentation Assays

In antigen presentation assays, T cells are cultured with antigenpresenting cells prepared according to the methods described above,under conditions that permit T cell recognition of peptides presented byMHC molecules on the antigen presenting cells. In some embodiments, Tcells are incubated with antigen presenting cells at 37° C. for between12-48 hours (e.g., for 24 hours). In some embodiments, T cells areincubated with antigen presenting cells at 37° C. for 3, 4, 5, 6, 7, or8 days. Numbers of antigen presenting cells and T cells can be varied.In some embodiments, the ratio of T cells to antigen presenting cells ina given assay is 1:10, 1:5, 1:2, 1:1, 2:1, 5:1, or 10:1. In someembodiments, antigen presenting cells are provided in an array (e.g., ina 96-well plate), wherein cells in each location of the array have beencontacted with sets of library cells, each set including a differentheterologous polypeptide. In certain embodiments, each location in thearray includes 1×10³ 1×10⁶ antigen presenting cells, and the cells arecontacted with 1×10³ 1×10⁶ T cells.

After T cells have been incubated with antigen presenting cells,cultures are assayed for stimulation. Lymphocyte stimulation can bedetected by any means known in the art. In some embodiments, culturesupernatants are harvested and assayed for secretion of a polypeptideassociated with activation, e.g., a cytokine, such as IFNγ, TNFα, TNFβinterleukin-2 (IL-2), IL-4, IL-5, IL-3, IL-10, IL-17, TGFβ, or GM-CSF.Cytokine secretion in culture supernatants can be detected, e.g., byELISA, bead array, e.g., with a Luminex® analyzer. Cytokine productioncan also be assayed by RT-PCR of mRNA isolated from the T cells, or byELISPOT analysis of cytokines released by the T cells. Otherpolypeptides associated with T cell activation, which may be assayed todetect stimulation, include perforin, granzyme, Fas ligand and CD40ligand, CD25, and CD69. In some embodiments, proliferation of T cells inthe cultures is determined (e.g., by detecting ³H thymidineincorporation). In some embodiments, target cell lysis is determined(e.g., by detecting T cell dependent lysis of antigen presenting cellslabeled with Na₂ ⁵¹CrO₄). Target cell lysis assays are typicallyperformed with CD8⁺ T cells. Protocols for these detection methods areknown. See, e.g., Current Protocols In Immunology, John E. Coligan etal. (eds), Wiley and Sons, New York, N.Y., 2007. One of skill in the artunderstands that appropriate controls are used in these detectionmethods, e.g., to adjust for non-antigen-specific background activation,to confirm the stimulatory capacity of antigen presenting cells, and toconfirm the viability of lymphocytes.

In some embodiments, antigen presentation assays are repeated usingantigen presenting cell and lymphocytes from different individuals,e.g., to identify antigens recognized by multiple individuals, orcompare reactivities that differ between individuals.

Applications

After it is determined that a heterologous polypeptide presented by anantigen presenting cell in an assay described herein stimulates humanlymphocytes, the heterologous polypeptide, now identified as an antigen,can be produced and incorporated into compositions for use in elicitingimmune responses. These and other applications are discussed below.

Human Cell Donors

One advantage of methods described herein is their ability to identifyclinically relevant human antigens. Humans that have been exposed to animmunogenic stimulus (e.g., from natural infection by an infectiousagent) contain lymphocytes that specifically recognize antigens of theinfectious agent, which are the product of an adaptive immune responsearising from the prior exposure. In some embodiments, these cells arepresent at a higher frequency than cells from a naive individual, and/orthe cells are readily reactivated when reexposed to the proper antigenicstimulus (e.g., the cells are “memory” cells). Thus, humans that havebeen exposed to an infectious agent are particularly useful donors ofcells for identifying antigens in vitro. The individual may be one whohas recovered from the infection, or one who has been exposed to theinfectious agent and remained asymptomatic. In some embodiments, theindividual has been recently infected with the infectious agent (e.g.,the individual was infected with the infectious agent less than threemonths, two months, one month, or two weeks, prior to isolation oflymphocytes and/or antigen presenting cells from the individual). Insome embodiments, the individual was first infected with the agent morethan three months, six months, or one year prior to isolation oflymphocytes and/or antigen presenting cells. In some embodiments, theindividual is latently or persistently infected with the infectiousagent. In some embodiments, the individual has been vaccinated againstan infectious agent of interest (e.g., with a whole cell bacterialvaccine, attenuated viral, peptide, or nucleic acid vaccine). Methodsherein may be used to analyze an immune response to a vaccine vectoritself. For example, cells of a patient immunized with a bacterial orviral vector that carries on or more antigens of a heterologous organismcan be isolated and analyzed to characterize an immune response againstthe bacterial or viral vector. In some embodiments, the cells from theindividual are screened in assays in which antigen presenting cells havebeen contacted with a library of cells whose members expresspolypeptides from the infectious agent, or a related species thereof.

In certain embodiments, cells from an individual who has been exposed toan infectious agent are used to identify antigens in a related species.In some embodiments, the infectious agent is a virus, and the relatedspecies is a virus in the same family. To give one example, cells from apatient infected with Epstein-Barr Virus (EBV) can be screened againstantigen presenting cells that have been incubated with a libraryexpressing polypeptides from Herpes Simplex Virus-2 (HSV-2).Polypeptides identified in such a screen may be useful as antigens thatelicit broadly cross-reactive immune responses protective againstmultiple types of herpes viruses.

In some embodiments, lymphocytes are screened against antigen presentingcells that have been contacted with a library of cells whose membersexpress polypeptides from an infectious agent, and the lymphocytes arefrom an individual who has not been exposed to the infectious agent. Insome embodiments, such lymphocytes are used to determine background(i.e., non-antigen-specific) reactivities. In some embodiments, suchlymphocytes are used to identify antigens, reactivity to which exists innaïve individuals.

Cells from multiple donors can be collected and assayed in methodsdescribed herein. In some embodiments, cells from multiple donors areassayed in order to verify that a given antigen is reactive in a broadportion of the population, or to identify multiple antigens that can belater combined to produce an immunogenic composition that will beeffective in a broad portion of the population.

The methods described herein have applications that extend beyondantigen identification. For example, the ability to rapidly detect humanantigen specific T cell responses in vitro can be used to determinewhether an individual has been exposed to an infectious agent or set ofinfectious agents. In some embodiments, antigen presentation assays areused to evaluate qualitative aspects of an individual's prior, orongoing, immunological reaction to an infectious agent. Some infectiousagents such as mycobacteria and hepatitis viruses cause persistentinfections. Other infectious agents are not eliminated effectively bythe immune response in patients. Comparing the types of antigenicreactivities associated with persistent infections and successfulimmunity to these agents can aid in the design of immunogeniccompositions that will be likely to provide protection. By way ofexample, in some embodiments, methods described herein are used tocompare antigenic reactivities found in patients suffering fromdisseminated herpes infections to those of patients in which a herpesinfection was resolved. These analyses may reveal antigenic reactivitiesprominent in resolved infections and lacking in disseminated infections.To provide another example, in some embodiments, methods herein are usedto compare antigenic reactivities in patients suffering from pelvicinflammatory disease (PID) due to an infectious agent such as C.trachomatis or N. gonorrhoeae to patients exposed to those agents, andthat do not present with pelvic inflammatory disease. The identificationof antigens associated with a particular pathological form of infectionfacilitates development of diagnostic assays to monitor the course ofinfection and determine the likelihood of developing the pathology. Forexample, antigens identified as associated with PID can be used indiagnostic assays for patients that are infected with a causative agent,and that have not yet developed PID, to help evaluate the likelihood ofoccurrence of this complication.

In still other, related embodiments, antigen presentation assays asdescribed herein are used to evaluate an individual's response to aninfectious agent to determine the stage of infection. For example, thepresence of reactivity to an antigen only expressed in a latent orpersistent phase of an infection can indicate that the infection hasprogressed to one of these stages. In some embodiments, antigenpresentation assays as described herein are used to evaluate responsesof subjects undergoing therapeutic treatment for an infection. In someembodiments, antigen presentation assays as described herein are used toevaluate responses of subjects who have been administered an immunogeniccomposition (e.g., as part of a clinical trial for a candidate vaccine).

Antigen presentation assays are useful in the context of non-infectiousdiseases as well. The methods described herein are applicable to anycontext in which a rapid evaluation of human cellular immunity isbeneficial. In some embodiments, antigenic reactivity to polypeptidesthat are differentially expressed by neoplastic cells (e.g., tumorcells) is evaluated. Sets of nucleic acids differentially expressed byneoplastic cells have been identified using established techniques suchas subtractive hybridization. Methods described herein can be used toidentify antigens that were functional in a subject in which ananti-tumor immune response occurred. In other embodiments, methods areused to evaluate whether a subject has lymphocytes that react to a tumorantigen or set of tumor antigens. In some embodiments, antigenpresentation assays are used to examine reactivity to autoantigens incells of an individual, e.g., an individual predisposed to, or sufferingfrom, an autoimmune condition. Such methods can be used to providediagnostic or prognostic indicators of the individual's disease state,or to identify autoantigens. For these assays, in some embodiments,libraries that include an array of human polypeptides are prepared. Insome embodiments, libraries that include polypeptides from infectiousagents which are suspected of eliciting cross-reactive responses toautoantigens are prepared. For examples of antigens from infectiousagents thought to elicit cross-reactive autoimmune responses, seeBarzilai et al., Curr Opin Rheumatol., 19(6):636-43, 2007; Ayada et al.,Ann N Y Acad Sci., 1108:594-602, 2007; Drouin et al., Mol Immunol.,45(1): 180-9, 2008; and Bach, J Autoimmun., 25 Suppl:74-80, 2005.

Epitope Identification

The present invention includes methods in which heterologouspolypeptides are expressed in library cells. After library cells areinternalized by antigen presenting cells, the heterologous polypeptidesare proteolytically processed within the antigen presenting cells, andpeptide fragments of the heterologous polypeptides are presented on MHCmolecules expressed in the antigen presenting cells. The identity of theheterologous polypeptide that stimulates a human lymphocyte in an assaydescribed herein can be determined from examination of the set oflibrary cells that were provided to the antigen presenting cells thatproduced the stimulation. In some embodiments, it is useful to map theepitope within the heterologous polypeptide which is bound by MHCmolecules to produce the observed stimulation. This epitope, or thelonger polypeptide from which it is derived (both of which are referredto as an “antigen” herein) can form the basis for an immunogeniccomposition, or for an antigenic stimulus in future antigen presentationassays.

Methods for identifying peptides bound by MHC molecules are known. Insome embodiments, epitopes are identified by generating deletion mutantsof the heterologous polypeptide and testing these for the ability tostimulate lymphocytes. Deletions which lose the ability to stimulatelymphocytes, when processed and presented by antigen presenting cells,have lost the peptide epitope. In some embodiments, epitopes areidentified by synthesizing peptides corresponding to portions of theheterologous polypeptide and testing the peptides for the ability tostimulate lymphocytes (e.g., in antigen presentation assays in whichantigen presenting cells are pulsed with the peptides). Other methodsfor identifying MHC bound peptides involve lysis of the antigenpresenting cells that include the antigenic peptide, affinitypurification of the MHC molecules from cell lysates, and subsequentelution and analysis of peptides from the MHC (Falk, K. et al. Nature351:290, 1991, and U.S. Pat. No. 5,989,565).

Immunogenic Compositions and Uses Thereof

The invention provides compositions that include an antigen or antigensidentified by methods described herein nucleic acids encoding theantigens, and methods of using the compositions. In some embodiments, acomposition includes antigens which are peptides 8-40 amino acids inlength (e.g., MHC binding peptides, e.g., peptides 8-25, 8-20, 8-15,8-12 amino acids in length). In some embodiments, a composition includesantigens which are polypeptides (e.g., polypeptides encoded by fulllength open reading frames of an infectious agent, or portions thereof).The compositions can include antigens which are, or which comprise, MHCclass I-binding peptides, MHC class II-binding peptides, or both MHCclass I and MHC class II-binding peptides. Compositions can include asingle antigen, or multiple antigens. In some embodiments, a compositionincludes a set of two, three, four, five, six, seven, eight, nine, ten,or more antigens. In some embodiments, the set of antigens is from asingle infectious agent. In some embodiments, the set of antigens isfrom two infectious agents.

The invention also provides nucleic acids encoding the antigens. Thenucleic acids can be used to produce expression vectors, e.g., forrecombinant production of the antigens, or for nucleic acid-basedadministration in vivo (e.g., DNA vaccination).

In some embodiments, antigens are used in diagnostic assays. For theseassays, compositions including the antigens can be provided in kits,e.g., for detecting antibody reactivity, or cellular reactivity, in asample from an individual.

In some embodiments, antigen compositions are used to induce an immuneresponse in a subject. In some embodiments, the subject is a human. Insome embodiments, the subject is a non-human animal. The antigencompositions can be used to raise antibodies (e.g., in a non-humananimal, such as a mouse, rat, hamster, or goat), e.g., for use indiagnostic assays, and for therapeutic applications. For an example of atherapeutic use, an antigen discovered by a method described herein maybe a potent T cell and B cell antigen, antibodies to which provideprotective immunity in vivo by neutralizing an infectious agent.Preparations of neutralizing antibodies may be produced by immunizing asubject with the antigen and isolating antiserum from the subject.Methods for eliciting high titers of high affinity, antigen specificantibodies, and for isolating the antigen specific antibodies fromantisera, are known in the art. In some embodiments, the antigencompositions are used to raise monoclonal antibodies, e.g., humanmonoclonal antibodies.

In some embodiments, an antigen composition is used to induce an immuneresponse in a human subject to provide protection from infection, or toprovide a therapeutic response. The protection can be complete orpartial protection. In some embodiments, an antigen composition elicitsan immune response to an infectious agent that causes the subject tohave milder symptoms and/or a shorter duration of illness, when exposedto the infectious agent, e.g., as compared to a subject that has notbeen administered the antigen composition. In some embodiments, where atherapeutic response is desired, the antigen composition elicits aresponse that reduces symptoms or duration of illness associated withthe infection, or reduced levels of the infectious agent, in thesubject, e.g., as compared to a subject that has not been administeredthe antigen composition.

In some embodiments, immunogenicity of an antigen is evaluated in vivo.In some embodiments, humoral responses to an antigen are evaluated(e.g., by detecting antibody titers to the administered antigen). Insome embodiments, cellular immune responses to an antigen are evaluated,e.g., by detecting the frequency of antigen-specific cells in a samplefrom the subject (e.g., by staining T cells from the subject withMHC/peptide tetramers containing the antigenic peptide, to detectantigen specific T cells, or by detecting antigen specific cells usingan antigen presentation assay such as an assay described herein). Insome embodiments, the ability of an antigen or antigens to elicitprotective or therapeutic immunity is evaluated in an animal model.

In some embodiments, the composition includes a pharmaceuticallyacceptable carrier or excipient. An immunogenic composition may alsoinclude an adjuvant for enhancing the immunogenicity of the formulation,(e.g., oil in water, incomplete Freund's adjuvant, aluminum phosphate,aluminum hydroxide, saponin adjuvants, or muramyl dipeptides). Otheradjuvants are known in the art.

In some embodiments, an immunogenic composition includes an antigenlinked to a carrier protein. Examples of carrier proteins include, e.g.,toxins and toxoids (chemical or genetic), which may or may not bemutant, such as anthrax toxin, PA and DNI (PharmAthene, Inc.),diphtheria toxoid (Massachusetts State Biological Labs; Serum Instituteof India, Ltd.) or CRM 197, tetanus toxin, tetanus toxoid (MassachusettsState Biological Labs; Serum Institute of India, Ltd.), tetanus toxinfragment Z, exotoxin A or mutants of exotoxin A of Pseudomonasaeruginosa, bacterial flagellin, pneumolysin, an outer membrane proteinof Neisseria meningitidis (strain available from the ATCC (American TypeCulture Collection, Manassas, Va.)), Pseudomonas aeruginosa Hcp1protein, E. coli heat labile enterotoxin, shiga-like toxin, human LTBprotein, a protein extract from whole bacterial cells, and any otherprotein that can be cross-linked by a linker. Other useful carrierproteins include bovine serum albumin (BSA), P40, and chickenriboflavin. Many carrier proteins are commercially available (e.g., fromSigma Aldrich.).

In some embodiments, an immunogenic composition including an antigenidentified by a method described herein is used in conjunction with anavailable vaccine. For example, an antigen identified as describedherein can be used as a supplemental component of a vaccine formulation,or as a boosting antigen in a vaccination protocol.

In some embodiments, an immunogenic composition is in a volume of about0.5 mL for subcutaneous injection, 0.1 mL for intradermal injection, or0.002-0.02 mL for percutaneous administration. A 0.5 ml dose of thecomposition may contain approximately 2-500 ug of the antigen.

In some embodiments an immunogenic composition is administeredparenterally (for instance, by subcutaneous, intramuscular, intravenous,or intradermal injection). In some embodiments, delivery by a means thatphysically penetrates the dermal layer is used (e.g., a needle, airgun,or abrasion).

In some embodiments, an immunogenic composition is administered to asubject, e.g., by intramuscular injection, intradermal injection, ortranscutaneous immunization with appropriate immune adjuvants.Compositions can be administered, one or more times, often including asecond administration designed to boost an immune response in a subject.The frequency and quantity of dosage of the composition can varydepending on the specific activity of the composition and can bedetermined by routine experimentation.

The formulations of immunogenic compositions can be provided inunit-dose or multi-dose containers, for example, sealed ampoules andvials and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use.

EXEMPLIFICATION Example 1: Preparation of a Library for Identificationof Human Antigens from Herpes Simplex Virus-2

Library Generation

An expression library was created from herpes simplex virus-2 (HSV-2)via gene-specific PCR amplification of each coding region, followed by asecond PCR reaction in which universal primers were used to append anadditional recombination sequence (the Gateway™ system) onto theamplified sequences for cloning. In some cases, gene targets weresynthesized by a commercial source and transferred within a plasmidvector.

PCR Primer Design

The open reading frames (ORFs) from the HSV-2 genome were generatedusing the published PubMed sequence under Accession No. NC_001798.Primers from all 77 genes were designed using the Primer3Plus softwareavailable at the following URL:bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi. The codingsequences for each gene were imported into the Primer3Plus software andthe software program calculated the primers based on set thresholds suchas Tm. Proteins predicted to have a signal sequence were truncated so asto be expressed without the signal sequence. The forward primers weredesigned starting at the transcriptional start site, ATG, and thereverse primers were upstream of the stop codon. A universal sequence5′-ACAAAAAAGCAGGCTGC-3′ (SEQ ID NO: ______) was added to the 5′ end ofeach forward primer and 5′-ACAAGAAAGCTGGGTAG-3′ (SEQ ID NO: ______) wasadded to the 5′ end of each reverse primer as an amplification site foraddition of attP cloning sequences, as described in the InvitrogenGateway Cloning manual.

PCR Amplification of Viral Genes

Each ORF was amplified in a 96-well plate with one ORF amplified perwell, using the primer sets described above, Herculase enhanced DNApolymerase (Stratagene, LaJolla, Calif.) and purified genomic DNA(Advanced Biotechnologies Inc., Columbia, Md.). The PCR cyclingparameters were 1 cycle at 98° C. for 4 minutes, followed by 30 cyclesof 98° C. for 20 seconds, 60° C. for 20 seconds, 72° C. for 30 seconds,and 1 cycle of 72° C. for 3 minutes 30 seconds. Five microliters of theresulting products were reamplified with primers5′-GGGACAAGTTTGTACAAAAAAGCAGGCTGC-3′ (SEQ ID NO: ______) and5′-GGGGACCACTTTGTACAAGAAAGCTGGGTAG-3′ (SEQ ID NO: ______) to add theattP cloning sequences to each product. The PCR cycling parameters weresame as the first round of PCR. PCR products were run on 1.2% E-gel(Invitrogen, Carlsbad, Calif.) to verify the size of each the PCRproducts.

Cloning of PCR Products

The amplicons generated for each open reading frame were individuallyrecombined into pDONR221 (Invitrogen) vector using BP Clonase II(Invitrogen) enzyme mix. Each reaction was incubated for 1 hour per kbof insert. All recombination reactions were transformed into Mach1chemically competent cells (Invitrogen) with heat shock. The resultingtransformant colonies were screened using colony PCR and consensusprimers that amplified a small fragment of the vector in addition to theinserted clone. For the PCR, 25 μl reactions [2×PCR Master Mix (Promega,Madison, Wis.), 6% DMSO (Fisher, Pittsburgh, Pa.), and M13 forward andreverse primers (10 μM)] were inoculated with a single transformant andheated to 95° C. for 4 minutes, followed by 25 cycles of (98° C. for 20seconds, 60° C. for 20 seconds, 72° C. for 1 minute per kb, and endingwith a final extension at 72° C. for 2 minutes. The reactions wereanalyzed by electrophoresis using either 1% 96-well or 1.2% 12-wellagarose E-Gels containing ethidium bromide and visualized under UVlight. Colonies positive for correct size inserts were cultured forplasmid DNA purification (Promega). After validation, purified plasmidDNA was subject to a second recombination reaction to recombine theinsert into pEXP4-DEST expression vector (Invitrogen) using LR ClonaseII enzyme mix (Invitrogen). Each reaction was incubated for 1 hour perkb of insert. All recombination reactions were transformed into Mach1chemically competent cells with heat shock.

The resulting transformant colonies were screened with using colony PCRtechniques with T7 consensus primers. For the PCR, 25 μl reactions wereinoculated with a single transformant in the same master mix describedabove and heated to 95° C. for 4 minutes, followed by 25 cycles of 98°C. for 20 seconds, 45° C. for 20 seconds, 72° C. for 1 minute per kb,and ending with a final extension at 72° C. for 2 minutes. The reactionswere analyzed by electrophoresis using either 1% 96-well or 1.2% 12-wellagarose E-Gels containing ethidium bromide and visualized under UVlight. Colonies (pEXP4-DEST(+) HSV-2 ORFs) positive for correct sizeinserts were cultured for plasmid DNA purification (Promega). Theplasmid DNA of each validated pEXP4-DEST(+) HSV-2 ORF was thentransformed with heat shock into Stbl2 chemically competent cells(Invitrogen) and also into Stbl2 expressing a cytoplasmic variant oflisteriolysin O (Stbl2/pAC(+)cLLO). Glycerol stocks were prepared fromeach of the libraries and stored at −80° C.

Synthetic Generation of Clones

Three open reading frames contained introns, and could not be PCRamplified due to an inavailablility of viral mRNA. As a result, thoseclones were generated de novo by a contract company (DNA 2.0, MenloPark, Calif.). In addition, a fourth clone was additionally synthesizedde novo after repeated unsuccessful attempts to clone it using standardmethods. The synthesized DNA fragments were received from DNA 2.0 aspDONR221 inserts after the fragments were size and sequence validated,and the inserts recombined into pEXP4-DEST as described above.

Library Sequence Validation

To verify the sequence of each cloned ORF, the entire pEXP4-DEST(+)ORFeome library was sent for single pass sequencing at Agencourt inBeverly, Mass. To prepare samples for sequencing, 200 μl media (10 g/Ltryptone and 5 g/L yeast extract (both from BD), plus 5 g/L sodiumchloride and 10% glycerol (both from Fisher) was aliquoted into a roundbottom, 96 well plate and each well was inoculated with a single clonefrom glycerol stocks of the Mach1/pEXP4(+) HSV-2 ORF library. Thecultures were grown statically for approximately 8 hours at 37° C., thenchecked for growth and frozen at −80° C. The plate was delivered toAgencourt via a courier service and single pass sequencing was done byprimer walking. Sequence data were analyzed using Vector NTI software(Invitrogen).

Library Induction

96-well deep-well plates containing were inoculated from the frozenlibraries by aliquoting 500 μL of LB+100 μg/mL carbenicillin (+40 μg/mLchloramphenicol if the library contained cLLO) per well of deep-well96-well plates, and with a 12-channel p200 pipet, gouging the tips intothe glycerol stock of frozen library and transferring the slurry to theLB. Plates were covered with a gas permeable membrane and incubatedovernight with shaking at 37° C. The next day, cultures wereback-diluted 1:100 into new plates in 1 mL of LB containing 0.2%maltose, 100 μg/mL carbenicillin (and 40 μg/mL chloramphenicol if thelibrary contained cLLO). Plates were covered with a gas permeablemembrane and incubated with shaking until the OD₆₀₀ reached between 0.6and 0.8. The median OD of the plate was calculated, and the averagenumber of bacteria per well were extrapolated. Ten millimolar MgSO₄ wasadded to each well followed by λCE6 bacteriophage (Novagen, Gibbstown,N.J.) at a multiplicity of infection of twelve phage to one bacterium.Plates were gently mixed then incubated at 37° C. without shaking for 20minutes then an additional hour and forty minutes with shaking, afterwhich the OD₆₀₀ was measured. From the OD calculations, the number ofbacteria per well were determined. Bacteria were pelleted andresuspended in 0.5% paraformaldehyde per well, incubated at roomtemperature for 30 minutes, and then washed two times with 1×PBS. Thebacteria were pelleted again, and then resuspended in RPMI containing10% FCS, 55 μM 2-mercaptoethanol, and 1× non-essential amino acids,sodium pyruvate, and L-glutamine for a final concentration of 2×10⁸bacteria per mL. The induced bacteria were aliquoted in 50μL volumesinto the same wells of new 96-well v-bottom plates; the plates werecovered with aluminum plate sealers, and frozen at −80° C.

Example 2: Screening a Library with Human Peripheral Blood Cells toIdentify Human Antigens from Herpes Simplex Virus-2

Preparation of PBMCs

Human whole blood from an HSV-2+/HSV-1-patient was collected viavenepuncture into eight sodium heparin CPT tubes (Becton Dickenson,Franklin Lakes, N.J.) at Bioreclamation Inc. (Hicksville, N.Y.), for atotal of 64 mL. The patient was diagnosed with in 1988, with a lastreported outbreak of April of 2007, and was on daily antiviral drugs.All tubes were centrifuged immediately after collection according tomanufacturer's instructions, and then shipped at 4° C. overnight. Uponreceipt of the tubes, the samples were acclimated to room temperature,mixed and then centrifuged again according to the manufacturer'sinstructions. Plasma was removed and frozen at −20° C., and the celllayer was transferred to a 50 mL centrifuge tube. The empty CPT tube waswashed three times with 3 mL PBS, and the washes were pooled into thetube containing the cells. Cells were pelleted and then resuspended indegassed PBS containing 2 mM EDTA and 0.5% BSA and counted.

Cell Sorting

Specific cell populations were positively selected out of the total PBMCusing magnetic beads (Miltenyi Biotec, Auburn, Calif.), following themanufacturer's instructions. CD4⁺ T cells were sorted first, followed byCD8⁺ T cells from the CD4 depleted fraction of cells. Finally, dendriticcells (DC) were sorted from the T cell depleted fraction. All sortedcells were stored on ice until used. A fraction of the remaining cells(T- and DC-depleted) were used to supplement the DCs for the ex vivoscreen of the library (described below) and the remainder were frozen ata concentration of 10⁶ cells/mL in characterized FBS containing 10% DMSOand stored in liquid nitrogen.

Ex Vivo Library Screening

The T cells (CD8⁺ for the cLLO-containing library and CD4⁺ for thenon-cLLO containing library) were counted and adjusted to aconcentration of 5×10⁵ cells/mL in complete medium (equal volumes ofRPMI-1640 and α-MEM with glutamax containing 10% human AB serum, 55M2-mercaptoethanol, and 1× each of HEPES, non-essential amino acids,penicillin/streptomycin, sodium pyruvate, and L-glutamine) containing 20U/mL rhIL-2 and returned to ice until use.

Plates containing the induced, fixed, and frozen libraries (with orwithout cLLO co-expressed) were removed from the −80° C. freezer andallowed to thaw at room temperature. Freshly isolated dendritic cellswere mixed with T- and DC-depleted cells for a final concentration of2×10⁶ cells/mL (the ratio of DC to non-DC/T cells was approximately 1:4)and 50 μL per well were loaded into round-bottom 96-well plates for atotal of 1×10⁵ APC per well. 50 μL of the thawed library was added toeach well (10⁷ bacteria per well), and the plates were incubated for 1 hat 37° C., 5% CO₂. Following incubation, the plates were washed threetimes with 1×PBS, centrifuging the plates with each wash to pellet thecells. The cells were then incubated in complete medium at 37° C., 5%CO₂ for an additional hour to allow antigen processing. Following thesecond incubation, the cells were pelleted and then fixed in 1%paraformaldehyde (PFA) for 15 minutes, after which they were washed andincubated with 120 mM lysine in PBS to remove any residual PFA.

After one more wash in PBS, the pellets were resuspended in 200 μL perwell properly diluted T cells. Plates were incubated at 37° C. for 24 h,after which 150 μL cell-free supernatant was harvested into the samewells of a new, v-bottom 96-well plate and frozen at −20° C. for ELISAanalysis. 150 μL fresh complete medium (no IL-2) was replaced in eachwell and the plates were allowed to incubate at 37° C., 5% CO₂ for anadditional 6 days, followed by a second harvest of 150 μL cell-freesupernatant for a second ELISA analysis.

Non-Specific Expansion of Sorted T Cells

Sorted T cells that weren't used in the ex vivo library screen describedbelow were non-specifically expanded using the Miltenyi MACSiBeadParticles following the recommended protocols. Briefly, 10⁸ anti-biotinMACSiBead particles were loaded with equal concentrations ofbiotin-conjugated CD2, CD3 and CD28-specific antibodies, and mixed withthe sorted T cells in complete medium at a ratio of 0.5:1. Cells andbeads were cultured at 37° C., 5% CO₂, at a density of 5×10⁶cells/mL/cm² for three days. On days three and five, the cultures weresplit into two equal parts and supplemented with medium containing 20U/mL rhIL-2. On day seven, the cells were used to screen the HSV-2library, as described below.

Library Screening with Non-Specifically Expanded T Cells

The library screen was set up as described for the ex vivo screen, withthe exceptions that medium alone or 5 g/mL PHA (Sigma) were added to Tcells in separate wells as negative and positive controls, respectively;the antigen presenting cells were T- and DC-depleted cells that had beenfrozen at −80° C. and thawed just prior to the assay; and thesupernatant from the assay was harvested and frozen only at 24 h.

ELISA Assay for IFNγ Determination

IFNγ levels in the supernatants were assayed using the OptEIA IFNγ ELISAkit from BD Biosciences (San Jose, Calif.) following the manufacturer'sinstructions with the exception that all volumes were halved.Supernatants were thawed at room temperature and diluted 1:5 and 1:20 inPBS containing 10% FCS. Samples that were positive in the ELISA wererescreened in a second ELISA assay to verify the results.

FIG. 1 shows results for CD8⁺ T cells freshly isolated from an HSV-2infected patient. Cells were cocultured with autologous antigenpresenting cells that had been pulsed with E. coli expressingcytoplasmic LLO. Cell free supernatants were harvested after 7 days ofculture. Each bar represents IFNγ levels secreted by T cells exposed toantigen presenting cells pulsed with a single viral clone. Black barsrepresent antigens that had been previously identified by others usingother methods. Six of the clones represented by white bars have no knownfunction, and likely would not have been identified by conventionalmethods of antigen discovery. These data show that methods describedherein successfully identify known human antigens to an infectiousagent, and can be used to reveal novel antigens.

Example 3. High-Throughput Library Validation of HeterologousPolypeptide Expression and MHC Presentation

In order to perform high-throughput determination of library proteinexpression and delivery to either the MHC class I or MHC class IIpresentation pathways, a C-terminal tag that contains both CD4⁺ and CD8⁺T cell epitopes from chicken ovalbumin (OVA₂₄₇₋₂₆₅ (PDEVSGLEQLESIINFEKL;SEQ ID NO: ______) and OVA₂₅₈₋₂₆₅ (SIINFEKL; SEQ ID NO: ______)respectfully, corresponding to positions in the amino acid sequencefound in GenBank® under Ace. No. NP_990483) was inserted into pDEST17 tocreate pDESTSL4.8. When PCR products containing ORFs (e.g., ORFs from aninfectious agent of interest) are recombined into pDESTSL4.8 using theGateway recombination system, the tag is fused to each expressed ORF onthe C-terminus. This tag can be used to determine whether each proteinwas expressed to full length and delivered to the proper MHCpresentation pathway.

For evaluating MHC class I presentation, the proteins are co-expressedwith cLLO in E. coli. The bacteria are then pulsed onto bone marrowmacrophages (BMM) derived from an H2^(b) haplotype mouse. The cLLOfacilitates delivery of each expressed ORF along with the C-terminal tagto the MHC class I presentation pathway in the BMM. If the full lengthof each ORF is expressed and properly delivered to the MHC class Ipathway, the OVA₂₅₈₋₂₆₅ peptide is processed and presented on the MHCclass I molecules, which are H2-K^(b) molecules, in the BMM cells.Presentation of this peptide can be detected through the use of B3Z Thybridoma cells, which activate upon recognizing this specific OVApeptide-MHC complex. Activated B3Z T cells produce β-galactosidase,which can be detected through the use of colorimetric β-galactosidasesubstrates such as chlorophenyl red β-D galactopyranoside (CPRG). CPRGchanges from a yellow to a dark magenta color when cleaved byβ-galactosidase, which indicates the ORF is expressed and processedproperly.

For MHC class II presentation, the proteins are induced in E. coli inthe absence of cLLO. The bacteria are then pulsed onto BMM derived froman H2^(k) haplotype mouse. The expressed protein remains in the vacuoleand is delivered to the MHC class 11 pathway along with the C-terminaltag. The OVA₂₄₇₋₂₆₅ tag is processed and presented on H2-A^(k) moleculeson the surface of the cell which are recognized by KZO T hybridomacells. KZO cells upregulate β-galactosidase in response to thepeptide-MHC complex which is again measured with CPRG detection. In thismanner, protein expression and both MHC class I and class IIpresentation can be confirmed prior to screening the library withpathogen-specific T cells.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description.Alternative methods and materials and additional applications will beapparent to one of skill in the art, and are intended to be includedwithin the following claims:

1-77. (canceled)
 78. A method of identifying a human subject exposed toan infectious agent, the method comprising: (a) providing a librarycomprising bacterial cells, wherein each bacterial cell of the librarycomprises a heterologous polypeptide encoded by an infectious agent; (b)contacting the bacterial cells with a first plurality of human cellswhich includes non-professional human antigen presenting cells thatinternalize the bacterial cells; (c) contacting the first plurality ofhuman cells with a second plurality of human cells from the humansubject which comprises human lymphocytes that are not selected forspecifically responding to an antigen, under conditions in whichlymphocytes are stimulated by polypeptides presented by the firstplurality of human cells; (d) determining whether a lymphocyte of thesecond plurality of human cells is stimulated by a heterologouspolypeptide presented by a cell of the first plurality of human cells;and (e) identifying the human subject as exposed to the infectious agentif a lymphocyte of the second plurality of human cells is stimulated bya polypeptide presented by a cell of the first plurality of human cells.79. The method of claim 78, wherein the infectious agent expresses afirst set of polypeptides during a first stage of infection and a secondset of polypeptides during a second stage of infection.
 80. The methodof claim 78, wherein the heterologous polypeptides comprise polypeptidesexpressed during the first stage of infection.
 81. The method of claim78, wherein the human subject is uninfected or asymptomatic.
 82. Amethod of identifying infection by an infectious agent in a humansubject, the method comprising: (a) providing a library comprisingbacterial cells, wherein each bacterial cell of the library comprises aheterologous polypeptide encoded by an infectious agent; (b) contactingthe bacterial cells with a first plurality of human cells which includesnon-professional human antigen presenting cells that internalize thebacterial cells; (c) contacting the first plurality of human cells witha second plurality of human cells from a first human subject whichcomprises human lymphocytes that are not selected for specificallyresponding to an antigen, under conditions in which lymphocytes arestimulated by polypeptides presented by the first plurality of humancells, wherein the first human subject is infected by the infectiousagent; (d) determining stimulation of a lymphocyte of the secondplurality of human cells by a heterologous polypeptide presented by acell of the first plurality of human cells; and (e) repeating steps (b)to (d), wherein the second plurality of human cells in step (c) are froma second human subject; wherein stimulation of a lymphocyte from thefirst human subject by a heterologous polypeptide and stimulation of alymphocyte from the second human subject by the heterologous polypeptideindicates the second human subject is infected by the infectious agent.83. The method of claim 82, wherein the second human subject isasymptomatic.
 84. The method of claim 82, wherein the first humansubject has a latent stage of infection.
 85. The method of claim 82,wherein the first human subject has a resolved infection.
 86. The methodof claim 82, wherein the second human subject is undergoing treatmentfor infection by the infectious agent.
 87. A method of identifying ahuman subject who has a cancer or tumor, the method comprising: (a)providing a library comprising bacterial cells, wherein each bacterialcell of the library comprises a heterologous polypeptide differentiallyexpressed in a cancer or tumor cell; (b) contacting the bacterial cellswith a first plurality of human cells which includes non-professionalhuman antigen presenting cells that internalize the bacterial cells; (c)contacting the first plurality of human cells with a second plurality ofhuman cells from the human subject which comprises human lymphocytesthat are not selected for specifically responding to an antigen, underconditions in which lymphocytes are stimulated by polypeptides presentedby the first plurality of human cells; (d) determining whether alymphocyte of the second plurality of human cells is stimulated by aheterologous polypeptide presented by a cell of the first plurality ofhuman cells; and (e) identifying the human subject as having a cancer ortumor if a lymphocyte of the second plurality of human cells isstimulated by a polypeptide presented by a cell of the first pluralityof human cells.
 88. A method of identifying a human subject who has anautoimmune condition, the method comprising: (a) providing a librarycomprising bacterial cells, wherein each bacterial cell of the librarycomprises a heterologous polypeptide expressed by a target cell ortissue of a human subject; (b) contacting the bacterial cells with afirst plurality of human cells which includes non-professional humanantigen presenting cells that internalize the bacterial cells; (c)contacting the first plurality of human cells with a second plurality ofhuman cells from the human subject which comprises human lymphocytesthat are not selected for specifically responding to an antigen, underconditions in which lymphocytes are stimulated by polypeptides presentedby the first plurality of human cells; (d) determining whether alymphocyte of the second plurality of human cells is stimulated by aheterologous polypeptide presented by a cell of the first plurality ofhuman cells; and (e) identifying the human subject as having anautoimmune condition if a lymphocyte of the second plurality of humancells is stimulated by a polypeptide presented by a cell of the firstplurality of human cells.
 89. A method of identifying an autoimmuneantigen, the method comprising: (a) providing a library comprisingbacterial cells, wherein each bacterial cell of the library comprises aheterologous polypeptide expressed in a target cell or tissue of a humansubject; (b) contacting the bacterial cells with a first plurality ofhuman cells which comprises human antigen presenting cells thatinternalize the bacterial cells; (c) contacting the first plurality ofhuman cells with a second plurality of human cells which comprises humanlymphocytes, under conditions in which lymphocytes are stimulated bypolypeptides presented by the first plurality of human cells; (d)determining whether a lymphocyte of the second plurality of human cellsis stimulated by a heterologous polypeptide presented by a cell of thefirst plurality of human cells, wherein stimulation of a lymphocyte ofthe second plurality of human cells by a heterologous polypeptidepresented by a cell of the first plurality of human cells indicates thatthe heterologous polypeptide is an autoimmune antigen.
 90. The method ofclaim 89, wherein the first plurality of human cells is isolated fromthe human subject.
 91. The method of claim 89, wherein the bacterialcells further comprise a cytolysin polypeptide.
 92. The method of claim91, wherein the cytolysin polypeptide is listeriolysin O (LLO).
 93. Themethod of claim 89, wherein the first plurality of human cells isprovided in an array, and wherein cells in each location of the arrayare contacted with a set of bacterial cells, each set comprising adifferent heterologous polypeptide.
 94. The method of claim 89, whereinthe first and second plurality of human cells comprise cells fromperipheral blood.
 95. The method of claim 89, wherein the firstplurality of human cells comprises dendritic cells.
 96. The method ofclaim 89, wherein the first plurality of human cells comprisesimmortalized cells.
 97. The method of claim 89, wherein the secondplurality of human cells comprises human lymphocytes derived from atarget cell or tissue of a human subject.
 98. The method of claim 89,further comprising formulating the antigen or a nucleic acid encodingthe antigen as an immunogenic composition.
 99. A method of identifyingan autoimmune antigen, the method comprising: (a) providing a librarycomprising bacterial cells, wherein each bacterial cell of the librarycomprises a heterologous polypeptide expressed in a target cell ortissue of a human subject; (b) contacting the bacterial cells with afirst plurality of human cells from one or more initial human subjectswhich comprises human antigen presenting cells that internalize thebacterial cells; (c) contacting the first plurality of human cells witha second plurality of human cells from the one or more initial humansubjects which comprises human lymphocytes, under conditions in whichlymphocytes are stimulated by polypeptides presented by the firstplurality of human cells; (d) determining whether a lymphocyte of thesecond plurality of human cells is stimulated by a heterologouspolypeptide presented by a cell of the first plurality of human cells,wherein stimulation of a lymphocyte of the second plurality of humancells by a heterologous polypeptide presented by a cell of the firstplurality of human cells indicates that the heterologous polypeptide isan autoimmune antigen; and (e) repeating steps (b) to (d) with humancells isolated from one or more additional human subjects, wherein theadditional human subjects have different clinical manifestations of anautoimmune disorder than the one or more initial human subjects. 100.The method of claim 99, wherein the first plurality of human cells isisolated from the human subject.
 101. The method of claim 99, whereinthe bacterial cells further comprise a cytolysin polypeptide.
 102. Themethod of claim 101, wherein the cytolysin polypeptide is listeriolysinO (LLO).
 103. The method of claim 99, wherein the first plurality ofhuman cells is provided in an array, and wherein cells in each locationof the array are contacted with a set of bacterial cells, each setcomprising a different heterologous polypeptide.
 104. The method ofclaim 99, wherein the first and second plurality of human cells comprisecells from peripheral blood.
 105. The method of claim 99, wherein thefirst plurality of human cells comprises dendritic cells.
 106. Themethod of claim 99, wherein the first plurality of human cells comprisesimmortalized cells.
 107. The method of claim 99, wherein the secondplurality of human cells comprises human lymphocytes derived from atarget cell or tissue of a human subject.
 108. The method of claim 99,further comprising formulating the antigen or a nucleic acid encodingthe antigen as an immunogenic composition.